SUMMARY Hematopoietic stem cells (HSCs), first produced in the developing vertebrate embryo, supply the lifelong foundation of the blood and immune systems. HSCs are therapeutically valuable as HSC transplantation (HSCT), the administration of donor HSCs to an immunocompromised recipient, is the standard of care for many hematological diseases. However, treatment availability remains problematic due to immune incompatibility and donor shortage. Likewise, while the number of transplanted HSCs is well known to directly impact engraftment efficiency, there are currently no established clinical protocols to successfully expand donor-harvested HSCs, nor to differentiate embryonic or induced pluripotent stem cells (iPSCs) into functional HSCs in vitro. Therefore, the identification of novel modifiers of de novo production of HSCs with long-term self-renewal and differentiation capacity is a major unmet clinical need. Despite more than a decade of research, current protocols rely primarily on enforced expression of transcription factors to help steer cells into an ?HSC-like? transcriptional program, however transplantation of these in vitro-derived HSCs into irradiated mice illustrates both limited long-term engraftment and multilineage potential. These observations imply that current in vitro differentiation strategies are missing critical cues which are essential to unlock or maintain full HSC potential in vivo. In the developing embryo, definitive HSCs arise from a unique population of mesodermal precursors termed hemogenic endothelium (HEC) through a process known as endothelial-to-hematopoietic transition (EHT). The transcription factor RUNX1, expressed in all sites of de novo HSC formation across vertebrates, is required for HSC specification and EHT. Our prior work revealed that Runx1 expression is strongly upregulated after initiation of the embryonic heartbeat, and HSC production is coordinated with the onset of vigorous circulatory flow and sheer stress. While mechanical properties of the niche, including sheer stress and circumferential stretch, are increasingly recognized as important stem cell cues in many contexts, the mechanism(s) by which mechanotransduction drives commitment to hemogenic fate and HSC productionduring vertebrate development remain largely unexplored. This study aims to characterize the role of biomechanical modulation of the hemogenic vascular niche in HSC formation in vivo and in vitro, with the overall goal of identifying the signaling pathway(s) connecting select biophysical forces to the gene regulatory network controlling HSC commitment. Our preliminary data indicate a novel, yet essential, role for circumferential stretch- stimulated activation of the transcription factor Yap1 in regulation of Runx1+ HEC specification and HSC production. Defining the molecular signaling pathways that mediate productive HSC formation in vivo will reveal new targets for optimizing the directed expansion and/or differentiation of adult-type HSCs for clinical use.